Glutaryl 7-ACA derivatives and processes for obtaining them

ABSTRACT

There are disclosed new compounds of formula (I), wherein R is: a hydrogen atom: a linear or branched C1-C4 alkyl group, unsubstituted or substituted by at least a phenyl group or at least a hydrogen atom: a benzyl group substituted by at least a linear or branched C1-C4 alkyl or alkoxy group or a nitro group; a silyl substituted by at least a linear or branched, unsubstituted or substituted C1-C4 alkyl group; n is 0 or 1; and Y is a radical of formula (II), wherein A is H, OH, Cl, CH2, CH2X, where X is F, Cl, Br, I, OH or OR&#39; and R&#39; is COCH3 or a linear or branched, unsubstituted or substituted C1-C4 alkyl group and . . . represents a single or a double bond, with the proviso that when n=0 and R is H, R&#39; is not a methyl group; and processes for obtaining them.

This is a §371 application of PCT/EP95/02802, filed Jul. 17, 1995.

The present invention relates to new cephalosporin intermediates,particularly to new 7-β-glutarylamido cephalosporins and to processesfor obtaining them, useful for the preparation of therapeutically,important cephalosporinic antibiotics.

It is well known that the major part of semisynthetic cephalosporins isobtained from 7-aminocephalosporanic acid (7-ACA) by acylation reactionsof the amino group in 7 and by nucleophilic substitution of the acetoxygroup in 3'. 7-ACA is still industrially produced by chemical hydrolysisof cephalosporin C through a complicated process which involves reagentsand solvents very toxic and polluting and extreme working conditions.

Enzymatic or chemical-enzymatic processes by glutaryl 7-ACA have beenlately developed to overcome these shortcomings. Said processes providetwo steps, in the first step cephalosporin C is transformedenzymatically in an aqueous medium in glutaryl 7-ACA, by oxidativedeamination with D-aminoacid oxidase (BE-A-736934 (Glaxo); JP-A-40588(Asahi); EP-A-0496993 (Antibioticos)!, or chemically, by oxidativetransamination U.S. Pat. No. 4079180 (Asahi)!. The glutaryl 7-ACA isthen deacylated by the glutaryl 7-ACA acylase enzyme JP-A-186599(Asahi); EP-A-0496993 (Antibioticos)!.

7-ACA cannot be used per se for the syntheses of the latestcephalosporins, for instance the 3-alkenyl-ones (Cefprozil, Cefdinir),and the quaternary 3'-ammoniummethyl ones (Cefepime), but it has to befirst undergone to protection reactions of the amino group (for instanceby acylation or transforming it in a Schiff's base) and of the carboxygroup (for instance by esterification). Besides, using 7-ACA isexpensive for preparing specific cephalosporins such as the3-cephem-3-halo substituted (for instance Cefaclor) or the unsubstitutedones (norcephalosporins, for instance Ceftizoxime and Ceftibuten), andthe industrial processes known hitherto for the production thereof use,as starting materials, compounds containing the penicillinic core, forinstance penicillin G or V, and come to obtaining the desired finalproducts by a complicated sequence of chemical reactions which mayprovide, subsequently, protecting of carboxyl, sulfoxidation, theopening of the penicillanic ring, the rearrangement to cephalosporanicring, etcetera. See, at this purpose, U.S. Pat. No. 4,052,387, U.S. Pat.No. 4,075,203, U.S. Pat. No. 4,081,440, U.S. Pat. No. 4,153,72, U.S.Pat. No. 4,031,084 and U.S. Pat. No. 4,346,218.

It is apparent that the glutaryl 7-ACA is not only cheaper than 7-ACAbut it shows a variety of advantages, from a chemical-synthetic point ofview, in respect with 7-ACA and cephalosporin C itself, the latter beingsometimes used to produce cephalosporinic antibiotics. Yet, isolatingglutaryl 7-ACA , given its high water solubility, is technically hardand expensive.

Other 7-ACA derivatives such as the known halomethyl derivatives, forinstance the 3-chloroismethyl or 3-bromomethyl derivatives, are obtainedfrom 7-ACA by a complicated sequence of protection reactions of theamino and carboxy groups, or by esters-sulfoxides of penicillin G bysophisticated technologies (opening of the penicillinic ring,electrochemical chlorinating, rearrangement to cephalosporinic ring).

Also the 3-exomethylene-derivatives of 7-ACA have a key structure forobtaining important cephalosporinic antibiotics. A few methods are knownfor transforming cephem derivatives into 3-exomethylene cephamderivatives.

These methods require the use of harmful and toxic compounds of Cr (II)(J. Chem. Soc. Chem. Comm. 800, 1972) or the utilize of electrochemicalreductions, expensive and technologically complicated (Torii et al,Bull. Chem. Soc. Jpn. 59, 3975, 1986) starting from 3-acetoxymethyl or3-halomethyl cephem, respectively.

There are other procedures which utilize Zn, as a reducing agent, yetstarting from more expensive 3-thio-functionalized derivatives.

The present invention relates to compounds having the following generalformula (I) ##STR1## wherein R is: a hydrogen atom;

a linear or branched C₁ -C₄ alkyl group, unsubstituted or substituted byat least a phenyl group or at least a hydrogen atom;

a benzyl group substituted by at least a linear or branched C₁ -C₄ alkylor alkoxy group or a nitro group;

a silyl substituted by at least a linear or branched, unsubstituted orsubstituted C₁ -C₄ alkyl group;

n=0 or 1; and Y is a radical of formula ##STR2##

wherein A is H, OH, Cl, CH₂, CH₂ X, where X is F, Cl, Br, I, OH or OR'and R' is COCH₃ or a linear or branched, unsubstituted or substituted C₁-C₄ alkyl group and . . . represents a single or a double bond, with theproviso that when n=0 and R is H, R' is not a methyl group.

The compounds of formula (I), as above defined, are new cephalosporinicintermediates useful for preparing cephalosporinic antibiotics.

In The compounds of formula (I) as above defined, wherein R is ahydrogen atom, a methyl, ethyl, propyl, t-butyl, 2,2,2-trichloroethyl,diphenylmethyl, 4-nitrobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl,trimethylsilyl and triethylsilyl group, are preferred.

The glutaric side chain is a protecting group very good for the 7-ACAamino group; in fact, on said chain, there is no group which caninterfere with the subsequent reactions, such as, for instance, theamino group of the aminoadipic chain of the cephalosporin C. Theglutaric side chain per se allows to stabilize the cephalosporinic coreand to carry out the functionalizing of the position 3 at best. It isgenerally necessary instead, to protect the amino group, in the case of7-ACA, to carry out the functionalizing of the position 3.

Besides, the absence of interfering groups on the glutaric chain greatlyhelps the chemical cut of said chain in the final stage of the processfor producing a certain antibiotic and, depending on the type of theantibiotic, said chain can be predictably removed, in a simpler way,enzymatically.

Another subject of the present invention is a process for obtaining thecompounds of formula (I), as above defined, wherein R and Y are as abovedefined, which comprises the enzymatic hydrolysis of the glutaryl 7-ACA,or the corresponding sulfoxide, in an aqueous solution, as it isobtained from the enzymatic reaction of the oxidative deamination ofcephalosporin C, for obtaining the corresponding desacetyl glutaryl7-aminocephalosporanic acid, the subsequent extractive esterification,and, optionally, the oxidation of the resulting diester, thus obtainingthe corresponding 3-cephem diester, or the diester-sulfoxide, of formula(I), as above defined, wherein A=CH₂ X, wherein X is OH or directlyesterifying the glutaryl 7-ACA, or the corresponding sulfoxide, thusobtaining the corresponding 3-cephem diester, or the diester-sulfoxide,of formula (I), as above defined, wherein A=CH₂ X, and X is OR' and R'is COCH₃ ; if desired, transforming the 3-hydroxymethyl cephemderivatives into the compounds of formula (I), wherein A=-CH₂ X and X isF, Cl, Br or I, by halogenating, working in an anhydrous environment; orX=-OR', wherein R' is a C₁ -C₄ alkyl, as above defined, by etherifying;the resulting halomethyl- and acetoxymethyl-derivatives, if desired, maybe transformed, respectively, by reductively dehalogenating anddeacylating, into the corresponding 3-exomethylene cepham derivatives,if desired, transforming the compounds of formula (I) so obtained intothe corresponding 3-hydroxy-3-cephem derivatives of formula (I), whereinA=OH, and n=0 or 1, by ozonolysis.

The following reaction schemes show some preferred examples of theprocess for obtaining the compounds of general formula (I).(DPM=diphenylmethyl). ##STR3##

Said diester of glutaryl 7-ACA may also be optionally reduced directlyto 3-exomethylene cepham as it is illustrated in the following reactionscheme which is preferred embodiment: ##STR4##

The enzymatic hydrolysis reaction is carried out on an aqueous solutionof the compound A in the presence of the acetylesterase enzyme,maintaining the pH between 7 and 8, at room temperature; the timenecessary to complete the reaction ranges between 80 minutes and 2hours.

The extractive esterification reaction is carried out directly on theaqueous solution of the desacetylglutaryl 7-ACA acid (B) coming from thepreceding step using diphenyldiazomethane dissolved in an organicsolvent, immiscible or poorly miscible in water, maintaining the pH ofthe mixture between 2.5 and 5 and the temperature between -10° C. and25° C.

Besides to diphenyldiazomethane (obtained for instance by oxydation ofthe benzophenone hydrazone), 4-nitrobenzylchloride,4-methoxy-benzylchloride, 4-nitrobenzylbromide, 4-methoxy-benzylbromideand the like, can be used as esterifying agents.

The organic solvents which can be used are ethyl acetate, methylacetate, toluene, methylene chloride, methylisobutylketone,methylethylketone and dimethylcarbonate. The time necessary to completethe reaction ranges between 4 and 12 hours.

The halogenation of the desacetylglutaryl diester (C) is carried out inan anhydrous solvent in the presence of a halogenating agent at a lowtemperature. Suitable halogenating agents can be phosphorous halogenidessuch as, for instance, phosphorous trichloride, phosphorouspentachloride, phosphorous tribromide, and thionyl chloride. Suitablesolvents can be dimethylcarbonate, methylene chloride and chloroform.

The temperatures range between -40° C. and 0° C., preferably -30° C. Thetime needed to complete the reaction ranges between 30 minutes and 2hours.

The reductive dehalogenation and deacylation of the halomethylglutaryldiester (D) and of the acetoxymethylglutaryl diesters (G and H),respectively, are generally carried out with zinc, activated before use,in the presence of a proton source and of a free base complexing agent,in an inert solvent, within a temperature range that goes between -50°C. and 0° C.

As a reducing metal, zinc in powder, activated by treatment with acidssuch as, for instance, 3% hydrogen chloride, is preferred, followed by awashing with water or with an organic solvent, preferably with thesolvent used for the reduction.

The solvents used are N,N-dimethylformamide, N,N-dimethylacetamide,N-methylformamide, dimethylsulfoxide.

Inorganic acids ammonium salts such as, for instance, ammonium chlorideand ammonium bromide, are used as the proton source.

The presence of ammonia into the reaction mixture causes theisomerization of 3-exomethylenecepham into 3-methylcephem; it istherefore convenient to add a free base complexing agent to the reactionmixture. The complexing agents mostly used are zinc chloride, zincbromide, ferrous trichloride.

The preferred temperature range goes from -50° C. to -30° C. The timeneeded to complete the reaction goes from 3 to 6 hours.

The ozonolysis reaction is carried out by passing ozone through asolution of the glutaryl 3-exomethylene cepham diester (E), or thecorresponding diester sulfoxide (N), in an inert solvent at atemperature between -80° C. and 0° C.

Suitable solvents are the ones in which the glutaryl exomethylene cephamdiesters are soluble.

The solvents used are methanol, ethanol, methyl acetate, ethyl acetate,methylene chloride, chloroform and mixtures thereof.

The preferred temperature range is between -80° C. and -50° C. Thedouble bond of the exomethylene derivative rapidly reacts with ozone toform an ozonide intermediate in situ. The ozone excess is removed makinga nitrogen or oxygen stream pass through the reaction mixture, when theozonide forming is complete. The ozonide is decomposed using reducingagents selective for the group such as, for instance, sodium bisolfite,sulfur dioxide, trimethylphosphite, triethylphosphite to give theglutaryl 3-hydroxy-3-cephem diester (F) and the corresponding diestersulfoxide (L). The reducing agent, used in excess, is added to thereaction mixture at a temperature between -80° C. and 0° C. Thesuspension is left under stirring until a negative response to the testpotassium iodide-soluble starch, is obtained.

It has to be noticed that the aqueous solution of the glutaryl 7-ACA, asit is obtained from the enzymatic reaction of oxidative deamination ofthe cephalosporin C, is directly used for obtainingglutarylcephalosporins which is the subject of the present invention.

In fact, to obtain the compounds object of the present invention, theisolation of the glutaryl 7-ACA is not necessary because the firstchanges which have to be effected to the molecule are carried out in anaqueous environment.

Besides, the organic solution of the glutaryl diester, obtained byextractive esterification, can be used as such for the subsequentreactions or it can be evaporated, obtaining the crystalline glutaryldiester which can be used for other reactions and in different solvents.

It has been further found that, differing from glutaryl 7-ACA anddesacetylglutaryl 7-ACA, the glutaryl diesters of general formula (I)are soluble in the organic solvents commonly employed in synthesizingthe cephalosporinic antibiotics, such as, for instance, methylenechloride, ethyl acetate, acetone, etc.

This peculiarity is extremely important from a synthetic point of view,particularly when reactions in an anhydrous environment have to becarried out.

The haloderivatives of formula (I) obtained carrying out the process ofthe invention, are particularly useful for obtaining variouscephalosporinic antibiotics such as, for instance, the ones of the lastgenerations, such as the 3-alkenyl cephalosporins, which are obtained bya Wittig reaction; the 3'-ammonium methyl cephalosporins, obtained byquaternising with heterocyclic bases or the 3-exomethylene cephamderivatives (intermediates for Cefaclor, 3-norcephalosporins, etc.),among which, the compounds of formula (I) wherein A=CH₂ obtained byreductive dehalogenation.

Also the 3-hydroxy derivatives, obtained by ozonolysis from theexomethylene derivatives of formula (I), carry to the core of Cefaclorby subsequent chlorination, or to the core of 3-norcephalosporins(Ceftibuten, Ceftizoxime, etc.) after eliminating the hydroxy group.

A further subject of the present invention is a process for obtainingthe 3-cephem derivatives of formula (I), as above defined, wherein A=Cl,which comprises the chlorinating of glutaryl 3-hydroxycephem diester offormula (I), as above defined, wherein A=OH, in the presence of anaprotic polar solvent, the hydrolysis of the two carboxy groups of theresulting glutaryl 3-chloro-3-cephem diester, in the presence of a Lewisacid, at a temperature comprised between the room temperature and 50° C.and the deacylation of the glutaryl 3-chloro-3-cephem-4-carboxylic acidso obtained.

The deacylation of said glutaryl 3-chloro-3-cephem-4-carboxylic acid maybe carried out both chemically and enzymatically to obtain thecorresponding 7-β-amino-3-chloro-3-cephem-4-carboxylic acid which, inits turn, may be suitably functionalized to obtain Cefaclor.

The following scheme shows a preferred embodiment of the inventiveprocess just above described. ##STR5##

The chlorination of the glutaryl 3-hydroxy-3-cephem diester (F) iscarried out in N,N-dimethylformamide in the presence of a chlorinatingagent at room temperature. As a chlorinating agent, phosphoroustrichloride and pentachloride, thionyl chloride, oxalyl chloride andfosgene may be mentioned.

It is preferable to use co-solvents such as dimethyl sulfoxide,tetrahydrofuran, dioxane, methylene chloride, dimethylacetamide. Thereaction is completed within 2-4 hours.

Generally, the chlorination of the glutaryl 3-hydroxy-3-cephem diesterof formula (I), as above defined, wherein A=OH, is also carried out inan anhydrous aprotic solvent, in the presence of atriarylphosphite-chlorine complex and of a base. Suitable solvents arechloroform, methylene chloride, and tetrahydrofuran.

The preferred chlorinating agent is the triarylphosphite-chlorinecomplex. Pyridine, quinoline and N,N-dimethylaniline are used as a base.The reaction is carried out within a temperature range going from -30°to +30° C. and preferably at -15° C.

The deprotection of the two carboxy groups of the glutaryl3-chloro-3-cephem diester (III) is carried out in anisole, in thepresence of a Lewis acid, heating at 30°-50° C. Among the suitable Lewisacids, there are aluminum trichloride, boron trifluoride andtrifluoroacetic acid. As a solvent, also phenol may be used.

The hydrolysis of the diesters may also be carried out in formic acidheating at 40°-50° C. The time needed to complete the reaction is 1-3hours.

The deacylation of the glutaryl 3-chloro-3-cephem-4-carboxylic acid (IV)may be carried out both chemically and enzymatically. The chemicaldeacylation (via iminochloride-iminoether) contemplates using ananhydrous inert solvent, an alkylchlorosilane, a base (scavenger of thedeveloping hydrogen chloride), a phosphorous halogenide and an alcohol,at a temperature between -50° C. and -15° C.

As a solvent, methylene chloride, chloroform and carbon tetrachloride,are used. The alkyl chlorosilanes used are dimethylchlorosilane,trimethylchlorosilane and triethylchlorosilane. As a base,dimethylamide, triethylamine, pyridine and N,N-dimethylaniline are used.Suitable halogenides are, for instance, phosphorous trichloride andpentachloride.

As an alcohol, methanol, ethanol, propanol, butanol and isobutanol, areused. The time needed to complete the reaction is 2-4 hours. Theenzymatic deacylation is carried out on an aqueous solution of thecompound IV, in the presence of an acylase enzyme such as, for instance,the glutaryl 7-ACA acylase, maintaining the pH between 7.5 and 9.0 atroom temperature. The time needed to complete the reaction is 30minutes-2 hours.

Another subject of the present invention is a process for obtaining the3-cephem derivatives of formula (I), as above defined, wherein A=Cl,which comprises the reduction of sulfoxide and the chlorination in asingle step, in the presence of an aprotic solvent, of the 3-cephemderivatives of formula (I), as above defined, wherein A=OH and n=1, togive the corresponding 3-chloro derivative. The compounds so obtainedcan be used for the production of important antibiotics by known methods(U.S. Pat. No. 3,925,372), particularly by the subsequent deprotectionin 7 and 4 to obtain the corresponding 7-β-amino-3-chloro-cephem esteras its halohydrate and the 7-β-amino-3-chloro-3-cephem-4-carboxylicacid, respectively.

The following reaction scheme shows a preferred embodiment of theprocess just above described. ##STR6##

If desired, the deprotection can also be effected simultaneouslyhalogenating in 3 and deprotecting in 7, thus obtaining thecorresponding 7-β-amino-3-chloro-3-cephem ester halohydrate and bysubsequent deprotection in 4, the corresponding7-β-amino-3-chloro-3-cephem-4-carboxylic acid.

The following reaction schemes show some preferred embodiments of theprocesses just above described. ##STR7##

The chlorination/reduction of the 3-hydroxy-3-cephem derivatives offormula (I), wherein n=1, is carried out in N,N-dimethylformamide in thepresence of a chlorinating/reducing agent at room temperature.

The preferred chlorinating/reducing agent is phosphorous trichloride.

It is possible to use cosolvents such as dimethylsulfoxide,tetrahydrofuran, dioxane, methylene chloride, dimethylacetamide. Thetime needed to complete the reaction is 2-4 hours.

The chlorination/reduction of the 3-hydroxy-3-cephem of formula (I),wherein n=1, is also carried out in an aprotic anhydrous solvent in thepresence of a triarylphosphite-chlorine complex and a base. Suitablesolvents are chloroform, methylene chloride and tetrahydrofuran. Thepreferred chlorinating agent is the triphenylphosphite-chlorine complex.As a base, pyridine, quinoline, and N,N-dimethylaniline, are used. Thereaction is carried out at a temperature range between -30° and +30° C.and preferably from -15° C. The reaction is completed in 0.5-2 hours.

A further subject of the present invention is a process for obtainingthe 3-hydroxy-3-cepham of formula (I), as above defined, wherein A=OHand n=0, which comprises the reduction of the glutaryl3-hydroxy-3-cephem diester of formula (F), as above defined, and, ifdesired, dehydrating the resulting 3-hydroxy-3-cepham derivative toobtain the corresponding 3-cephem of formula (I), as above defined,wherein A=H and n=0.

The following reaction scheme shows a preferred example of the processjust above described. ##STR8##

The following examples are merely meant to illustrate the presentinvention, yet not limiting it in any way. The ¹ H-NMR spectra wererecorded by a 300 MHz Brucker spectrometer.

The abbreviations used in the description of the ¹ H-NMR spectra mean:s=singlet; d=doublet; dd=double doublet; t=triplet, q=quadruplet;m=multiplet; brs=broad singlet. The chemical shifts are reported in ppm.

The MS spectra were recorded by a spectrometer VARIAN MAT 311 A. Ozonegenerator: Fisher OZON 502. All of the syntheses steps were followedkinetically by HPLC chromatographic analysis using a Shimadzu HPLCLC-10AD endowed with a Brownlee RP18 column, a Shimadzu SPD-6A detectorand a Shimadzu C-R4A integrator.

EXAMPLE 1

Preparation of the diphenylmethyl 7-β-(4-diphenylmethoxycarbonyl)-butanamido!-3-chloro-3-cephem-4-carboxylate(III).

6.73 g of phosphorous trichloride (0.048 mol) were slowly added to acooled solution (water/ice bath) of 13.25 g of the compound (F), (0.02mol) in 110 ml of N,N-dimethylformamide, maintaining the temperaturelower than 5° C.

The transformation of the product (F) is followed by HPLC. The reactionmixture was left under stirring at room temperature, for 4 hours andthen poured in 75 ml of an aqueous solution of hydrogen chloride at 5%,maintaining the temperature lower than 10° C.

The suspension was extracted with ethyl acetate (2×100 ml) and thecollected organic extracts were rinsed with an aqueous solution of 5%hydrogen chloride (50 ml), followed by a buffer at pH 6,5 (100 ml). Theorganic phase was dried (Na₂ SO₄) and the solvent was evaporated todryness under reduced pressure.

The title product, recovered from the oily residue by crystallisationwith methanol, was a slightly yellow chrystalline powder (8.60 g, 0.12mol) having a HPLC purity of 95%. The molar yield, calculated on thebasis of the HPLC title, was 60% on the recovered product.

¹ H-NMR (CDCl₃, 300 MHz)

1.97(m, 2H, --CH₂ --); 2.24 (t, 2H, --CH₂ --); 2.48 (t, 2H, --CH₂);3.41-3,71 (ABq, 2H, J=17.0 Hz, C2); 4.98 (d, 1H, J=5.0 Hz, C6); 5.81(dd,1H, J=5.0-9.0 Hz, C7) 6.45 (d, 1H, J=9.0 Hz, NH); 6.89-6.99 (s, 2H,diphenylmethyl-CH); 7.23-7.45 (brs, 20 H, aromatic).

Elemental analysis for the product (III) C₃₈ H₃₃ N₂ O₆ ClS: calculated(%): C, 67.00; H, 4.88; N, 4.11; Cl, 5.20; S, 4.70 found (%): C, 66.66;H, 5.07, N, 4.08; Cl, 4.80; S, 4.66.

The mass spectrum ("Field Desorption" technique) showed a molecular peakat 680, which agrees with the molecular weight calculated for C₃₈ H₃₃ N₂O₆ ClS.

EXAMPLE 2

Preparation of the7-β-(4-carboxybutanamido)-3-chloro-3-cephem-4-carboxylic acid (IV).

A solution of 14.34 g of the product (III) (0.2 mol, HPLC purity 95%) in200 ml of anisole was added to a solution of 6.49 g of anhydrousaluminum trichloride (0.047 mol) in 80 ml of anisole, maintaining thetemperature lower then 30°-35° C. for 2 hours. The transformation of theproduct (III) was followed by HPLC technique. The reaction mixture,after cooling at -10° C., was poured into 100 ml of a cool solution of2% HCl, maintaining the temperature lower than 15° C. The aqueous acidphase was saturated with sodium chloride and extracted with ethylacetate (3×100 ml). The collected organic extracts were concentrated byevaporating the solvent till the crystallisation of the product IV wasobtained.

The title product was a slightly yellow, microcrystalline solid (5.50 g,0.015 mol) having a HPLC purity higher than 95%. The molar yield,calculated on the basis of the HPLC title, was 75% on the recoveredproduct.

¹ H-NMR (DMSO, 300 MHz)

1.72 (m, 2H, --CH₂ --); 2.22 (m, 4H, --CH₂ --); 3.69-3.98 (ABq, 2H,J=18.0 Hz, C2); 5.18(d, 1H, J=4.6 Hz, C6); 5.67 (dd, 1H, J=4.6-8.0 Hz,C7); 8.95 (d, 1H, J=8.0 Hz, NH). Analysis-Calculated for C₁₂ H₁₃ ClN₂ O₆S: C, 41.33; H, 3.76; Cl, 10.16; N, 8.03. Found: C, 41.80; H, 3.89; Cl,9.80; N, 7.79.

The mass spectrum (FAB mass spectrum) showed the protonated molecularion at m/z 349, which agrees with the molecular weight calculated forC₁₂ H₁₃ ClN₂ O₆ S.

EXAMPLE 3

Preparation of the7-β-(4-carboxybutanamido)-3-chloro-3-cephem-4-carboxylic acid (IV).

A solution of 14.34 g of the product (III) (0.02 mol, HPLC purity 95%)in 70 ml of 99% formic acid was heated at 45° C. for 30 minutes. Theacid excess was removed by evaporating at the same temperature. Theproduct (IV) was then recovered from the residual oil by crystallisationfrom ethyl acetate.

The product was a slightly yellow microcrystalline solid (5.58 g, 0.016mol) having a HPLC purity higher than 95%. The molar yield, calculatedon the basis of the HPLC title, was 80% on the recovered product.

¹ H-NMR (DMSO, 300 MHz)

1.72 (m, 2H, --CH₂ --); 2.22 (m, 4H, --CH₂ --); 3.69-3.98 (ABq, 2H,J=18.0 Hz, C2); 5.18(d, 1H, J=4.6 Hz, C6); 5.67 (dd, 1H, J=4.6-8.0 Hz,C7); 8.95 (d, 1H, J=8.0 Hz, NH).

EXAMPLE 4

Preparation of the 7-β-amino-3-chloro-3-cephem-4-carboxylic acid (V)(Chemical method)

To a suspension of 7.34 g of the product (IV) (0.02 mol, HPLC purity95%) in 60 ml of anhydrous methylene chloride, cooled at 0° C., 9.10 gof N,N-dimethylaniline and 5.43 g of trimethyl chlorosilane were added,maintaining the temperature lower than 30° C. 5.20 g of phosphorouspentachloride (0.025 mol) were added to the solution cooled at -5° C. 16ml of isobutanol were added very slowly maintaining the temperaturelower than -20° C., after having maintained the reaction mixture understirring for two hours at -40° C. 30 ml of water were added after anhour maintaining the temperature lower than 0° C. and the pH at thevalue of 7 by concentrated ammonia. The organic phase was removed andthe product recovered from the aqueous phase by precipitation at theisoelectrical point (pH 3.9-4.0) using concentrated HCl to lower the pH.The precipitated solid was filtered, washed with methanol and dried.

The microcrystalline solid was recognized as the title product. Themolar yield, calculated on the basis of the HPLC title, was 85% based onthe recovered product. The data of the NMR spectra of the obtainedproduct were coincident with the ones known in literature.

EXAMPLE 5

Preparation of the 7-β-amino-3-chloro-3-cephem-4-carboxylic acid (V)Enzymatic method!

One liter of an aqueous solution of 25 mM KH₂ PO₄ containing 15.0 g ofthe product (IV) (0.043 mol) was brought at pH 8 with 2% NH₄ OH. Thesolution was thermostated.

The solution was thermostated at 25° C. and added with 10 g of theglutaryl 7-ACA acylase enzyme immobilized on a resin. The mixture waslet under stirring for an hour maintaining the pH at the value of 8 byadding 2% NH₄ OH. The enzyme was filtered and the product was recoveredfrom the aqueous phase by precipitation at the isoelectrical point (pH3.9-4.0), using concentrated HCl to lower the pH. The precipitated solidwas filtered, washed with methanol and dried.

The microcrystalline solid was recognized as the title product. Themolar yield, calculated on the basis of the HPLC title, was 90% on therecovered product. The data of the NMR spectrum of the resulting productwere coincident with the ones known in literature.

EXAMPLE 6

Preparation of diphenylmethyl 7-β-(4-diphenylmethoxycarbonyl)-butanamido!-3-hydroxy-cepham-4-carboxylate(IX).

A solution of 13,6 g of the product (F) (0.02 mol), 8.2 ml of glacialacetic acid, 50 ml of methanol and 150 ml of toluene, was cooled at -55°C., added with 1,85 g of sodium boron hydride (0.048 mol), thetemperature was raisen up to -30° C. soon after the end of the addition.The cold reaction mixture was poured into 145 ml of a saturated solutionof NaHCO₃, maintaining the temperature lower than 15° C. The organicphase was concentrated by evaporating the solvent till thecrystallisation of the product (IX) was obtained.

The title product was a white microcrystalline solid (8.86 g, 0.012 mol)having a HPLC purity higher than 90%. The molar yield, calculated on thebasis of the HPLC title, was 60% on the recovered product.

¹ H-NMR (DMSO, 300 MHz)

1.96 (m, 2H, --CH₂ --); 2.23 (t, 2H, --CH₂ --); 2.48 (t, 2H, --CH₂ --);2.61-2.98 (AB of ABX, 2H, JAB=13.8 Hz, JBX=10.0 Hz, JAX=3.5 Hz, C2),(3.32 (d, 1H, J=7.8 Hz, OH); 4.08 (m, 1H, J=10.0-7.8-6.0 Hz, C3); 4.84(d, 1H, J=6.0 Hz, C2); 5.07 (d, 1H, J=4.0 Hz, C6); 5.53 (dd, 1H,J=9.0-4.0 Hz, C7); 6.51 (d, 1H, J=9.0 Hz, NH); 6.87-6.92 (s, 2H,diphenylmethyl-CH); 7.23-7.40 (brs, 20 H, aromatic).

EXAMPLE 7

Preparation of diphenylmethyl 7-β-(4-diphenylmethoxycarbonyl)-butanamido!-3-acetoxymethyl-3-cephem-4-carboxylate(G).

The procedure described below consists of extractively esterifying 7-β-(carboxybutanamido!-3-acetoxymethyl-3-cephem-4-carboxylic acid andisolating the corresponding title diphenylmethyl ester. 29.0 g of anethyl acetate solution of diphenyldiazomethane were added to 1 l anaqueous solution of 25 mM KH₂ PO₄ containing 23 g of the7-β-(4-carboxybutanamido)-3-acetoxymethyl-3-cephem-4-carboxylic acid(0.058 mol). The biphasic mixture was put under stirring, thermostatingat 10° C. 18% HCl was slowly added dropwise till pH=3.5, maintainingsuch pH for 1.5 hours and then lowering it at pH=2.5, maintaining understirring for 30 minutes. The phases were separated when the synthesisended, the organic phase being treated with 500 ml of H₂ O and bringingat pH=7,5 by Na₂ CO₃.

The phases were separated by rinsing the organic phase two times with500 ml of a saturated solution of NaCl. The organic phase was dried withNa₂ SO₄ and the solvent was eliminated under vacuum (40 torr) at 30° C.The oily crude product was collected with 300 ml of isopropanol andunder stirring for 1 hour at room temperature. The product was recoveredby filtration and undergone to a further purification treating it with250 ml of 1,1-diethoxymethane and under stirring for 30 minutes at 25°C.

26.2 g of a white solid recognized as the title product, having a HPLCpurity of 94%, were obtained.

¹ H-NMR (CDCl₃ -200 MHz)

2,02 ppm (s, 3H, CH₃ CO); 1,90-2,10(m,2H--CH₂ --); 2,26 ppm (t, 2H,--CH₂ --); 2,52 ppm (t, 2H--CH₂ --); 3,33 3,55 (ABq, 2H, J=18,6 Hz,C-2); 4,79-5,04 ppm (ABq, 2H, J=13,6 Hz, CH₂ --OCOCH₃); 4,79 ppm (d, 1H,J=5 Hz, C-6); 5,85 ppm (dd, 1H, J=5,0-8,8 Hz C-7); 6,09 ppm (d, 1H,J=8,8 Hz, --NH); 6,90 ppm (s, 1H, -diphenylmethyl-CH); 6,96 ppm (s, 1H,diphenylmethyl-CH); 7,31-7,46 ppm (m, 20H aromatic).

EXAMPLE 8

Preparation of diphenylmethyl 7-β-(4-diphenylmethoxycarbonyl)-butanamido!-3-acetoxymethyl-3-cephem-4-carboxylate-1-oxide(H). 25 g (0.031 mol) of the product (G) were dissolved in 300 ml ofanhydrous CH₂ Cl₂ maintaining the temperature at 18°-20° C. 7,1 ml of a32% solution of peracetic acid were dropwise added within 5 minutes inthe flask, maintaining the solution under stirring. The disappearance ofthe compound (G) was observed in HPLC after 15 minutes. The solvent waseliminated under vacuum (80 torr) and the crude residue was collectedwith 300 ml of ethyl acetate.

The resulting solution was washed two times with 300 ml of 8% NaHCO₃solution: The phases were separated. Counterextracting with 200 ml ofAcOEt was effected from the aqueous phase. The phases were separated.The organic phases were reunited and washed 2 times with 400 ml of asaturated solution of NaCl. The organic phase was concentrated to 250 mland left at 0° C. After 8 hours the product was filtered and dried undervacuum at 30° C. for 4 hours.

21,1 g of the title product having a HPLC purity of 96% were recovered.

¹ H-NMR (CDCl₃, 200 MHz)

2,03 ppm (s, 3H, CH₃ CO); 1,97-2,08 ppm (m, 2H, --CH₂ --); 2,31 ppm (t,2H, --CH₂ --); 2,52 (t, 2H, --CH₂ --); 3,20-3,80 (ABq, 2H J=19,0 Hz,C-2); 4,46 ppm (d, 1H, J=4,8 Hz, C-6); 4,74-5,32 ppm (ABq, 2H, J=14,5Hz,--CH₂ --OCOCH₃); 6,11 ppm (dd, 1H, J=4,8 Hz, 9,9 Hz, C-7); 6,66 ppm(d, 1H, J=9,9 Hz, NH); 6,89 ppm ((s, 1H, diphenylmethyl-CH); 6,96 ppm(s, 1H, diphenylmethyl-CH); 7,26-7,50 ppm (m, 20H, aromatic).

EXAMPLE 9

Preparation of diphenylmethyl 7-β-(4-diphenylmethoxycarbonyl)-butanamido!-3-methylene-cepham-4-carboxylate-1-oxide(N)

10 g of the (0.014 mol) of the compound (H) were dissolved in 100 ml ofabsolute DMF. This solution was dropwise added within 2 minutes to aflask containing a suspension of 18 g of activated Zn in powder, 30 g ofNH₄ Cl, 20 g of ZnCl₂ in 100 ml of absolute dimethylformamide, cooled at0° C. Vigorous stirring was maintained for 6 hours at 0° C. Thesuspension was filtered and the solid residue was washed on the filterwith 200 ml of ethyl acetate. The ethyl acetate used for washing wasmixed with the filtrate obtaining the precipitation of an inorganicresidue. The inorganic residue was filtered and 450 ml of H₂ O wereadded to the resulting solution under stirring for 30 minutes. Afterfiltering the biphasic mixture, the phases were separated and theorganic phase was rinsed 2 times with 250 ml of a 18% solution of NaCl.Drying with Na₂ SO₄, eliminating the solvent and crystallising the oilycrude product in 200 ml of methanol leaving under stirring for 4 hoursat room temperature and at 5° C. for 3 hours was effected.

The filtered product was dried in an oven under vacuum at 30° C. 6 g ofa white solid, recognized as the title product, were recovered. HPLCpurity=98.1%

¹ H-NMR (CDCl₃ 200 MHz)

1;92-2,04 (m, 2H,--CH₂ --); 2,25 ppm (t, 2H,--CH₂ --); 2,48 ppm (t, 2H,--CH₂ --); 3,36-3,62 ppm (Abq, 2H, C-2); 4,80 ppm (d, 1H, J=4,8 Hz, C-6)5,32 ppm (s, 1H, C=CH₂); 5,42 (s, 1H, C=CH₂); 5,77 ppm (s, 1H, C-4);5,92 ppm (dd, 1H, J=4,8 Hz-10,1 Hz, C-7); 6,85 ppm (d, 1H, J=10,1 Hz,NH); 6,83 ppm (s, 1H diphenylmethyl-CH); 6,87 ppm (s, 1H,diphenylmethyl-CH); 7,22-7,39(m, 20H, aromatic).

EXAMPLE 10

Preparation of diphenylmethyl 7-β-(4-diphenylmethoxycarbonyl)-butanamido!-3-hydroxy-3-cephem-4-carboxylate-1-oxide(L).

In a solution of 13.54 g of the product N (0.02 mol) in 150 ml ofethylacetate, cooled at -65° C., ozone was insufflated for 25 minutes(about 1,3 mmol 0₃ /min).

The excess of ozone was removed insufflating oxygen for 5 minutes andnitrogen for 15 minutes. 7 ml of triethylphosphite (0.04 mol) were thenadded, maintaining the temperature lower than -50° C. At the end of theaddition the solution gave a negative response to the peroxide test(KI-soluble starch).

The reaction mixture was poured in 50 ml of an aqueous solution of 5%HCl, after having allowed the temperature to raise up to -25° C., andleft for 30 minutes under vigorous stirring at 15°-20° C. The organicphase was rinsed with 5% aqueous solution of sodium chloride (2×100 ml),dried with sodium sulfate and, after evaporating the solvent theresidual oil was solidified by treatment with n-pentane.

The amorphous solid obtained was recognized as the title product. Themolar yield, calculated on the basis of the HPLC title was 90% on therecovered product.

¹ H-NMR (CDCl₃, 300 MHz)

2.02 (t, 2H, --CH₂ --); 2.32 (t, 2H, --CH₂ --); 2.52 (t, 2H, --CH₂ --);3,41÷3.72 (ABq, 2H, J=19.0÷1.0 Hz, C₂); 4.52 (dd, 1H, J=4.0÷1.0 Hz, C₆);6.03 (dd, 1H, J=4.0÷10.0 Hz, C₇); 6.66 (d, 1H, J=10.0 Hz, NH); 6.89÷6.92(s, 2H, diphenylmethyl-CH); 7.20÷7.40 (brs, 20 H, aromatic); 11.70 (s,1H, OH).

EXAMPLE 11

Preparation of the diphenylmethyl 7-β-(diphenylmethoxycarbonyl)-butanamido!-3-methylene-cepham-4-carboxylate(E).

A solution of 10 g of the product (G) (0.012 mol) in 100 ml of absolutedimethylformamide was added to a suspension of 15 g of activated Zn inpowder, 12 g of NH₄ Cl and 15 g of ZnCl₂ in 100 ml of absolutedimethylformamide cooled to 0° C. After 4 hours under vigorous stirringat 0° C. the suspension was filtered and the solid rinsed with 200 ml ofethyl acetate which were added to the filtrate. Filtering was effectedagain to eliminate the precipitated inorganic solid and 400 ml of H₂ Owere added to the resulting solution stirring for 30 minutes at roomtemperature.

The biphasic solution was filtered and, after separating, the organicphase was rinsed two times with 200 ml of a 18% solution of NaCl anddried over Na₂ SO₄. The solvent was evaporated and the resulting oil waspowdered from n-pentane. 6,2 g of light yellow solid recognized as thetitle product. HPLC purity=90%.

EXAMPLE 12

Preparation of diphenylmethyl 7-β-(diphenylmethoxycarbonyl)-butanamido!-3-chloro-3-cephem-4-carboxylate(III)

9.81 g of phosphorous trichloride (0.070 mol) were slowly added to acooled solution (water bath/ice) of 13.54 g of the compound L (0.02 mol)in 110 ml of N,N-dimethylformamide, maintaining the temperature lowerthan -55° C. The reaction mixture was left under stirring at -55° C. forone hour and at room temperature for 4 hours; then it was poured in 75ml of an aqueous solution of 5% hydrogen chloride, maintaining thetemperature lower than 10° C. The suspension was extracted with ethylacetate (2×100 ml) and the collected organic extracts were rinsed withan aqueous solution of 5% hydrogen chloride (50 ml) followed by a bufferat pH 6.5 (100 ml). The organic phase was dried (Na₂ SO₄), and thesolvent evaporated to dryness under reduced pressure.

The title product, recovered from the oily residue by crystallisationwith methanol, was a light yellow crystalline solid (6.81 g, 0.012 mol)having a HPLC purity of 95%. The molar yield, calculated on the basis ofthe HPLC title, was 50% on the recovered product.

¹ H-NMR (CDCl₃, 300 MHz)

1,97 (m, 2H, --CH₂ --); 2,24 (t, 2H,--CH₂ --); 2,48 (t, 2H, --CH₂ --);3,41-3,71 (Abq, 2H, J=17,0 Hz, C2); 4,98 (d, 1H, J=5,0 Hz, C6); 5,81(dd, 1H, J=3,0-9,0 Hz, C7); 6,45 (d, 1H, J=9,0 Hz, NH); 6,89-6,99 (s,2H, diphenylmethyl-CH); 7,23-7,45 (bis, 20H, aromatic).

EXAMPLE 13

Preparation of the diphenylmethyl7-β-amino-3-chloro-3-cephem-4-carboxylate hydrochloride (VIII)

2,4 ml of pyridine and 6,0 g of phosphorous pentachloride were added toa solution of 13.62 g of the product III (0.020 mol) in 90 ml ofmethylene chloride, cooled at -15° C. The reaction mixture was cooled at-30° C. after having left it under stirring at -15° C., and added with19 ml of isobutanol.

The title product was precipitated from the reaction mixture, broughtback to -15° C., and left under stirring. The precipitated solid wasfiltered, rinsed with methylene chloride and dried. The obtained productwas a white microcrystalline solid (6.12 g 0.014 mol); the ¹ H-NMR datawere coincident with the ones of the true product, as reported inliterature.

The molar yield, calculated on the basis of the HPLC title, was 70% onthe recovered product.

EXAMPLE 14

Preparation of the 7-β-amino-3-chloro-3-cephem-4-carboxylic acid (V).

A solution of 8.75 g of the product VIII (0.02 mol) in 20 ml of 99%formic acid, was heated at 54° C. for 30 minutes. The acid excess wasremoved by evaporating at the same temperature, the residual oil wasdispersed in 30 ml of water and the aqueous phase was rinsed with 30 mlof ethyl acetate (2×15 ml).

The product was recovered by precipitating at the isoelectrical point(pH 3,9-4,0) using concentrated ammonia to raise the pH. Theprecipitated solid was filtered, rinsed with methanol and dried. Theproduct was a slightly yellow, microcrystalline solid (3.94 g, 0.0168mol); the ¹ H-NMR data were coincident with the ones of the authenticproduct reported in literature.

EXAMPLE 15

Preparation of the diphenylmethyl7-β-amino-3-chloro-3-cephem-4-carboxylate hydrochloride (VIII)

Chlorine gas was insufflated in a solution of 13,10 mltriphenylphosphite (0.0685 mol) in 90 ml of methylene chloride, cooledat -15° C., until a permanent yellow solution was obtained.

The chlorine excess was then eliminated by adding 0.4 ml oftriphenylphosphite (0.0015 mol). A solution of 13.58 g of the product L(0.02 mol) in 15 ml of methylene chloride was added to the obtainedsolution containing about 0.070 mol of the triphenylphosphite-chlorinecomplex. A solution of 5.5 ml of pyridine was then slowly added thereto.19 ml of isobutanol was then slowly added after having left the reactionmixture under stirring for an hour at -15° C.

HCl gas was insufflated into the mixture after an hour from the end ofthe addition obtaining the precipitation of the title product. Theprecipitated solid was filtered, rinsed with methylene chloride anddried.

The obtained product was a white microcystalline solid (7.0 g, 0.016ml); the ¹ H-NMR data were coincident to the ones of the true productreported in literature. The molar yield, calculated on the basis of theHPLC title, was 80% on the recovered product.

EXAMPLE 16

Preparation of the diphenylmethyl7-β-amino-3-chloro-3-cephem-4-carboxylate hydrochloride (VIII)

Chlorine gas was insufflated in a solution of 13,10 ml oftriphenylphosphite (0.0485 mol) in 90 ml of methylene chloride, cooledat -15° C. until a permanent yellow solution was obtained. The chlorineexcess was then eliminated by adding 0.4 ml of triphenylphosphite(0.0015 mol). A solution of 13,26 g of the product F (0.002 mol) in 15ml of methylene chloride was added to the solution obtained containingabout, 0.0050 mol of the triphenylphosphite-chlorine complex.

A solution of 4.0 ml of pyridine in 16 ml of methylene chloride was thenslowly added thereto. 19 ml of isobutanol were slowly added after havingleft the reaction mixture under stirring for an hour at -15° C. HCl gaswas insufflated into the mixture after an hour from the end of theaddition, obtaining the precipitation of the title product. Theprecipitated solid was filtered, rinsed with methylene chloride anddried.

The product obtained was a white microcrystalline solid (7.4 g, 0.017mol); the ¹ H-NMR data were coincident to the ones of the true productas reported in literature. The molar yield calculated on the basis ofthe HPLC tittle was 85% on the recovered product. EXAMPLE 17

Preparation of the7-β-(4-carboxybutanamido)-3-hydroxymethyl-3-cephem-4-carboxylic acid (B)

1 l of a 25 mM aqueous solution of KH₂ PO₄ containing 25 g of the7-β-(4-carboxybutanamido)-3-acetoxymethyl-3-cephem-4-carboxylic acid (A)(0,065 mol) was carried to pH=7.5 by KOH.

Thermostating at 20° C. and adding 75 g of the acetylesterase enzymesupported on a resin, were effected.

The admixture was put under stirring for 45 minutes maintaining at pH=7by adding a solution of 7% NH₄ OH.

The enzyme was filtered and the solution carried at pH=6.5 with 12% HCl.

A solution containing 21.7 g of the compound B was obtained.

The yield in the solution, calculated by HPLC, was 97%.

The title product was not isolated because the solution was used in thenext step.

EXAMPLE 18

Preparation of the diphenylmethyl 7-β-(4-diphenylmethoxycarbonyl)-butanamido!-3-hydroxymethyl-3-cephem-4-carboxylate(C).

The procedure described below comprises the extractive esterification ofthe 7-β-(4-carboxybutanamido)-3-hydroxymethyl-3-cephem-4-carboxylic acid(B) and the isolation of the corresponding title diphenylmethyl ester.36.9 g of diphenyldiazomethane in ethyl acetate solution were added tothe solution containing 21,7 g of the compound B (0.063 mol).

The biphasic mixture was put under stirring thermostating at 10° C.

18% HCl was slowly added dropwise till pH=2-2.5, carrying on for 4hours, maintaining the pH constant.

After completing the synthesis, the phases were separated, the organicphase being treated with 500 ml of H₂ O, and bringing the pH at 6.5 withNa₂ CO₃. The phases were separated rinsing the organic phase two timeswith a NaCl saturated solution. The organic phase was dried with Na₂ SO₄and the solvent was eliminated under vacuum (40 torr) at 20° C.

The oily crude product was purified by treatment with a 1:9 ethylacetate/n-pentane mixture.

25.7 g of a white solid recognized as the title product, having a HPLCpurity of 90%, were obtained. ¹ H-NMR (CDCl₃, 200 MHz) 2.00 (m,2H,--CH₂--);2.25 (t, 2H, --CH₂ --); 2.5 (t, 2H, --CH₂ --); 3.55 (s, 2H, C-2);3.95÷4.4 (ABq, 2H, J=12,8 Hz, CH₂ --OH); 4.9 (d, 1H, J=4.8 Hz, C-6); 5.9(dd, 1H, J=4.8÷8.8 Hz, C-7); 6.2 (d, 1H, J=8.8 Hz, NH); 6.9 (s, 1H,diphenylmethyl-CH); 6.94 (s, 1H, diphenylmethyl-CH); 7.2÷7.4 (brs, 20H,aromatic).

EXAMPLE 19

Preparation of the diphenylmethyl 7-β-(4-diphenylmethoxycarbonyl)-butanamido!-3-chloromethyl-3-cephem-4-carboxylate(D).

10 g (0.015 mol) of the compound C obtained in the foregoing examplewere dissolved in 70 ml of anhydrous CH₂ Cl₂, lowering the temperatureat -30° C. 3.9 g (0.02 mol) of PCl₅ were inserted in the flask. Thedisappearing of the compound C was observed by HPLC after 30 minutes.2.6 ml (0.02 mol) of triethylamine were dropwise added, leaving understirring for 30 minutes. The reaction was estinguished with 77 ml of H₂O.

The phases were separated, and washing of the organic phase for twotimes with a NaCl saturated solution, was effected.

The solvent was eliminated under vacuum after having been dried on Na₂SO₄. The crude product was purified with cyclohexane. 9.2 g of the titleproduct were recovered.

The molar yield was 94% on the basis of the HPLC title. ¹ H-NMR (CDCl₃,200 MHz) 2.00 (m, 2H,--CH₂ --); 2.25 (t, 2H,--CH₂ --); 2.5 (t, 2H,--CH₂--); 3.42÷3.52 (ABq, 2H, J=18.7 Hz, C-2); 4.4 (s, 2H, CH₂ --Cl); 5.00(d, 1H, J=4.8 Hz, C-6); 5.85 (dd, 1H, J=4.8÷8.9 Hz,C-7); 6.2 (d, 1H,J=8.9 Hz, NH); 6.9 (s, 1H, diphenylmethyl-CH); 7.00 (s, 1H,diphenylmethyl-CH); 7.2÷7.5 (brs, 20H, aromatic).

EXAMPLE 20

Preparation of the diphenylmethyl 7-β-(4-diphenylmethoxycarbonyl)-butanamido!-3-methylene-cepham-4-carboxylate(E).

A solution of 6.94 g of the compound D obtained in the foregoing example(0.01 mol) in 80 ml of absolute, was added to a suspension of 12.5 g ofactivated Zn in powder, 10 g of NH₄ Cl and 7 g of ZnCl₂ in 80 ml ofabsolute dimethylformamide cooled at -45° C.

After 3 hours under vigorous stirring at -40° C., the suspension wasfiltered and the solid washed with 100 ml of ethyl acetate which wereadded to the filtrate.

200 ml of an aqueous solution of 18% NaCl were added to the resultingsolution, stirring for 30 minutes at room temperature. The organic phasewas subsequently washed for two times with 100 ml of an aqueous solutionof 18% NaCl, dried with Na₂ SO₄, evaporating the solvent andcrystallizing the resulting oil from CH₂ Cl₂ /n-pentane.

4.6 g of a light yellow solid, recognized as the title product, wereobtained. HPLC purity=90%

NMR (CDCl₃, 200 MHz) 1.98 (m, 2H, --CH₂ --); 2.23 (t, 2H, --CH₂ --); 2.5(t, 2H, --CH₂ --); 3.09÷3.50 (ABq, 2H, J=13, 9Hz,C-2); 5.21÷5.24 (s, 2H,C₃ =CH₂); 5.32 (s, 1H, C-4); 5.35 (d,1H, J=4.3 Hz,C-6); 5.64 (dd, 1H,J=4.3÷9.2 Hz, C-7); 6.10 (d, 1H, J=9.2 Hz, NH); 6.86÷6.88 (s, 2H,diphenylmethyl-CH); 7.23÷7.37 (brs, 20H, aromatic).

EXAMPLE 21

Preparation of the diphenylmethyl 7-β-(4-diphenylmethoxycarbonyl)-butanamido!-3-hydroxy-3-cephem-4-carboxylate(F).

Ozone (0.75 mmol 0.3/min) was insufflated for about 20 minutes into asolution of 6.60 g of the compound E obtained in the foregoing example(0.01 mol) in 160 ml of CH₂ Cl₂ and 40 ml of CH₃ OH, cooled at -75° C.After said time, the reaction mixture developed a light blue colour. Theozone excess was removed insufflating O₂ for 5 minutes and N₂ for 15minutes.

19 g of sodium metabisulfite were added thereto, the mixture beingstirred for 30 minutes at -75° C. and for about an hour at 0° C. Aftersaid time, the suspension gave a negative response to the peroxide test(KI-soluble starch).

The liquid phase was decanted and rinsed with water.

The organic phase, after drying with Na₂ SO₄ and evaporating thesolvent, gave an amorphous solid recognized as the title product. Purity(HPLC)=80%.

¹ H-NMR (CDCl₃, 200MHz) 2,00 (m,2H,--CH₂ --); 2.28 (t,2H,--CH₂ --); 2.52(t, 2H,--CH₂ --); 3.27÷3.48 (ABq, 2H, J=13.9 Hz, C-2); 5.01 (d, 1H,J=4.5 Hz, C-6); 5.71 (dd, 1H, J=4.5÷8.5 Hz, C-7); 6.26 (d, 1H, J=8.5 Hz,NH); 6.87÷6.89 (s, 2H, diphenylmethyl-CH); 7.23÷7,37 (brs, 20H,aromatic).

We claim:
 1. The compound of formula: ##STR9##
 2. The compound offormula: ##STR10##
 3. The compound of formula: ##STR11##
 4. The compoundof formula: ##STR12##
 5. The compound of formula: ##STR13##